An often recurring problem for the researcher, whether he be working in the university or industry, is the need to remove one or only a few specific substances from a solution admixed with a large variety of other solutes of no interest. In some cases, the desired component(s) may be known beforehand, and the objective is one of isolation and purification of desired quantities. A problem of this type is presented by the isolation of opioid alkaloids from the milky exudate of the poppy plant. In other cases, one may be faced with unknowns as regards the number, as well as the identity, of the solutes of interest, and the objective is to ascertain both. Identification of the urinary metabolites of an experimental drug would illustrate the problem in the second type of cases.
There are certain inherent difficulties in the problem under consideration which have defied the development of a simple and satisfactory method applicable in most such cases. The commonalities underlying the problem at hand are: (1) presence of water; (2) presence of inorganic salts; and (3) the multitude and diversity of compounds present with dissimilar and similar properties (chemical and physical) to the solute(s) of interest. In addition, not infrequently the task of selective isolation is further exacerbated by extremely low concentrations of those solutes relative to totally dissolved material.
In contrast, most methods of separation depend upon the availability of narrowly defined classes of purified components as starting materials. As examples constituting such classes may be mentioned amino acids, carbohydrates, lipids, etc. The presence of other substances, with differing physical and/or chemical properties, may preclude the employment of separation techniques otherwise appropriate to the task. For example, water present in an incompletely dried lipid sample would deactivate silicic acid (choice of adsorbent for chromatography of lipids), resulting in loss of adsorption and separation of components. Should the components of interest be present in very low concentrations, a prepurification (enrichment) step may be required, if for no other reason than to prevent overloading the system of separation employed, while trying to achieve detectable levels. For example, drug levels in body fluid are often too low to permit their direct use for GLC, even if the various other components present did not interfere with the conditions of analysis.
For the reasons mentioned above, it is, therefore, not surprising that no general one-step procedure has been developed, applicable to and capable of resolving complex aqueous solutions as characterized in the foregoing. Usually a multistep procedure is employed. The particular approach chosen is usually determined by the nature of the solutes of interest.
A common starting step is multiple solvent extraction of the sample. Often the pH is adjusted for preferential partitioning which further may be enhanced by additions of strong electrolytes. When dealing with unknowns, extractions are carried out at three pH ranges; that is, acid, neutral and alkaline. When extractions by solvents as described do not prove satisfactory, the sample is subjected to chemical or enzymatic hydrolysis, followed by reextraction. Continued failure of extraction at this point would necessitate recourse to other methods.
Some of the salient drawbacks associated with solvent extraction are the following. To obtain a relatively clean and near complete extract of the desired solute(s) is more of an event in serendipity than a virtue of the method. Usually the extract contains some of the desired solute(s), but the bulk is extraneous matter. Moreover, solutes of interest present in the extract reflect not so much their proportional concentrations in the sample, but rather their partition coefficients under the conditions of extraction. Unless the latter is known to start with, no conclusion can be drawn as to the quantitative distribution of the solutes of interest in the sample. If the desired components are of semipolar or polar character, extraction is usually incomplete, resulting not only in waste of material, but also in the need for larger sample size.
The process of extraction often is difficult to perform. Especially solutions of biological origin, when shaken with nonmiscible solvents, tend to form emulsions. This may be so severe as to prevent the applicability of the method or may require additional drastic measures. Finally, the extracts so obtained often require extensive further purification.
Ion-exchange resins also are employed for the selective isolation of solutes. By suitable manipulation of pH and buffer strength, one may obtain enriched fractions of the desired components. Here again there are certain difficulties. Use of buffers may increase the electrolyte content in the eluates, requiring additional procedures for their removal. Strong ion-exchange resins may act as strong acids or bases, resulting in hydrolysis or chemical breakdown of sensitive materials. They are stable only to varying degrees, and subunits of their matrix can contaminate the effluents. The resins, apart from the functional groups they possess, may act as nonspecific adsorbents and thus introduce an additional variable.
In view of the lack of methodology capable of resolving complex biological fluids into their components or at least into narrow classes with only a few constituents, efforts were directed at total analysis in one step. Earlier, Dalgliesh (Biochem. J., 1966, 101, 792) extracted whole urine under drastic conditions, treated the residue with a variety of derivatizing reagents and analyzed the resultant derivatives by GLC. More recently, Thompson (Res. Comm. Chem. Path. and Pharm., 1977, 16, 145) went a step further by attempting GLC analysis of total urinary residue without any preliminary purification or separation.
Some of the obvious drawbacks of such an approach immediately should be apparent: (a) unknown metabolites may escape detection, since there is no universal derivatizing reagents; (b) such analyses are time-consuming, due to the large number of compounds that do get derivatized; (c) column life is greatly shortened by injection of salts and other underivatizeable compounds; and (d) perhaps most importantly the chromatograms so obtained often cannot be interpreted meaningfully. A glance at such a chromatogram would reveal that the density of information provided is so great as to preclude both quantitative and qualitative assessment of individual components. Frequent partial or complete overlaps obscure detection of components, and the range of concentrations usually encountered exceed the linearity of the detection systems employed. This complicated picture further may be confused by the likely presence of spurious peaks, due to the well-known tendency of some of the commonly used derivatizing reagents to produce multiple derivatives from single compounds.